The present invention relates to nucleic acid sequences and methods useful for producing recombinant human soluble epoxide hydrolase (sEH).
Epoxide hydrolases (EC 3.3.2.3) are a family of enzymes which hydrolyze a variety of exogenous and endogenous epoxides to their corresponding diols. Epoxide hydrolases have been found in tissues of all mammalian species tested. The highest levels of the enzyme were found in liver and kidney cells (see Wixtrom and Hammock Pharmacology and Toxicology (Zakim, D. and Vessey, D. A., ed.)1:1-93, Wiley, New York, 1985).
Four principal EH's are known, leukotriene epoxide hydrolase, cholesterol epoxide hydrolase, microsomal EH and sEH (previously called cEH). The leukotriene EH acts on leukotriene A.sub.4, whereas the cholesterol EH hydrates compounds related to the 5,6-epoxide of cholesterol (Nashed, N. T., et al., Arch. Biochem. Biophysics., 241:149-162, 1985; Finley, B. and B. D. Hammock, Biochem. Pharmacol., 37:3169-3175,1988). The microsomal EH hydrates monosubstituted, 1,1-disubstituted, cis-1,2-disubstituted epoxides and epoxides on cyclic systems. The more abundant soluble EH hydrates a wide range of epoxides not on cyclic systems. The following schematic illustrates the hydrolysis of an epoxide to yield the vicinal product as catalyzed by sEH. ##STR1##
Compounds containing the epoxide functionality have become common environmental contaminants because of their wide use as pesticides, sterilants, and industrial precursors. Such compounds also occur as products, by-products, or intermediates in normal metabolism and as the result of spontaneous oxidation of membrane lipids (i.e. see, Brash, et.al., Proc. Natl. Acad. Sci., 85:3382-3386 (1988), and Sevanian, A., et.al., Molecular Basis of Environmental Toxicology (Bhatnager, R. S., ed.) pp. 213-228, Ann Algor Science, Michigan (1980), which are incorporated herein by reference). As three-membered cyclic ethers, epoxides are often very reactive and have been found to be cytotoxic, mutagenic and carcinogenic (i.e. see Sugiyama, S., et.al., Life Sci. 40:225-231 (1987), which is incorporated herein by reference). Cleavage of the ether bond in the presence of electrophiles often results in adduct formation. As a result, epoxides have been implicated as the proximate toxin or mutagen for a large number of xenobiotics.
Soluble epoxide hydrolase as well as microsomal epoxide hydrolase metabolize a wide range of epoxides to their corresponding diols. Because of their broad substrate specificity these two enzymes are thought to play a significant role in ameliorating epoxide toxicity. Reactions of detoxification typically decrease the hydrophobicity of a compound, resulting in a more polar and thereby excretable substance. Soluble EH in human lymphocytes decreases the induction of sister chromatid exchanges by trans-.beta.-ethylstyrene, an in vitro substrate of sEH (i.e. see Kramer, A., et.al., Biochem. Pharmacol. 42:2147-2152 (1991), incorporated herein by reference). Also, sEH of rodent decreased the mutagenicity of epoxide-containing compounds in the Ames' Salmonella assay (i.e. see E1-Tantawy, M. A. and B. D. Hammock, Mut. Res. 79:59-71, which is incorporated herein by reference).
In addition to degradation of potential toxic epoxides, sEH are believed to play a role in the formation or degradation of endogenous chemical mediators. For instance, cytochrome P450 epoxygenase catalyzes NADPH-dependent enatioselective epoxidation of arachidonic acid to four optically active cis-epoxyeicosantrienoic acids (EETs) (Karara, A., et al., J. Biol. Chem., 264:19822-19877, (1989)). Soluble epoxide hydrolase has been shown in vivo to convert these compounds with regio- and enantiofacial specificity to the corresponding vic-dihydroxyeicosatrienoic acids (DHETs). Both liver and lung cytosolic fraction hydrolyzed 14,15-EET, 8,9-EET and 11,12-EET in that order of preference. Purified sEH selected 8S,9R- and 14R,15S-EET over their enantiomers as substrates. Studies have revealed that EETs and their corresponding DHETs exhibit a wide range of biological activities. Some of these activities include involvements in luteinizing hormone-releasing hormone, stimulation of luteinizing hormone release, inhibition of Na.sup.+ /K.sup.+ ATPase, vasodilation of coronary artery, mobilization of Ca.sup.2+ and inhibition of platelet aggregation. Soluble epoxide hydrolase is believed to play a role in these biological activities by contributing to the regulation of the steady state levels of EETs and DHETs.
The sEH protein from human, rhesus monkey, baboon, rabbit, rat and mouse liver was affinity purified by Silva, M. H. and B. D. Hammock, Comp. Biochem. Physiol. 87B:95-102 (1987). The cDNA for human microsomal epoxide hydrolase has been cloned and expressed in COS-1 cells by Skoda, R. C., et. al., J. Biol. Chem., 263:1549-1554 (1988). Although sEH of mouse, rat, and human have similar molecular weight and immunoreactivity as shown by Dietze, E. C., et. al., Int. J. Biochem., 22:461-470 (1990), the data of Meijer, J. and J. W. Depierre, Chem.-Biol. Interact., 64:207-249 (1988), suggest sEH of primates and non-primate species differ in substrate specificity and inhibition.